Time-series analysis of transcription factor knockdown in Drosophila S2R+ cells using RNA-Seq.. Drosophila melanogaster

To understand how RNAi depletion of transcription factors impact the Drosophila transcriptome, we performed selective knockdown of 46 transcription factors in Drosophila S2R+ cell line. We treated cells with long double strand RNAs by bathing for 1 to 3 days. We purified polyA+ RNA and prepared strand-specific RNA-Seq libraries. We quantitatively measured transcriptomic responses using a time series analysis. Overall design: We depleted a total of 46 different transcription factors in S2R+ cells. In each well of a 96 well plate, we targeted a single transcription factor for knockdown using a single double stranded RNA reagent. Except for two transcription factors that we have used on only one reagent (alphaCOP and Trf), a total of two different double strand RNAs reagents were used per transcription factor. We had six different control wells in our plates: two wells targeting the E. coli LacZ gene, one well targeting GFP, one well targeting thread (= Death-associated inhibitor of apoptosis 1. Flybase ID: FBgn0260635) gene -- which gave us a visual information of knockdown by causing cell death, and two wells where we didn’t treat any double strand RNA. Total number of these samples and controls are 96. We prepared three of such plates, and incubated for 24, 48, and 72 hours. From the cells, we isolated polyA+ RNA to prepare strand-specific RNA-Seq libraries. We performed RNA-Seq based on Illumina-platform to have read lengths of 76 base pairs, in single-ends.